Long chain fatty acids appear to be transported by Escherichia coli via a translocation catalyzed by an acyl CoA synthetase. This hypothesis was supported by the findings that (1) the synthetase was partially membrane bound; (2) uptake and the synthetase had similar temperature dependencies; (3) mutants lacking the synthetase did not take up long chain fatty acids; (4) uptake was inhibited in cells treated with compounds which interfere with ATP synthesis or the cycling of CoA. Short chain fatty acid transport has been studied in isolated membrane vesicles prepared from an ato R mutant of E. coli. Vesicles prepared from such mutants, which synthesize an acetyl CoA:butyrate CoA transferase, took up butyrate and uptake was dependent on added acetyl CoA. Kinetic and chemical properties of the uptake system and the CoA transferase were virtually indistinguishable. Virtually all the radioactivity taken up by the vesicles was identified as butyryl CoA and this observation and kinetic studies strongly suggest that the CoA transferase translocates rather than traps butyrate.